Electron induced fluorescent method for nucleic acid sequencing

ABSTRACT

An apparatus, compositions and related methods for sequencing a target nucleic acid are described. In certain embodiments, the apparatus is a microfluidic apparatus comprising an input chamber, microchannel, output chamber and a detection unit that is operatively connected to the microchannel. In preferred embodiments, the methods include hybridizing a target nucleic acid to one or more probe libraries, moving the hybridized target nucleic acid past the detector, and detecting bound probes. Probe libraries may comprise oligonucleotides or oligonucleotide analogs, preferably with each probe uniquely labeled. A linear order of labeled probes hybridized to the target nucleic acid can be detected and the target nucleic acid sequence deduced. In preferred embodiments, probe labels are detected by analysis of electron-induced fluorescence of probes labeled with conductive polymers.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the fields of molecular biologyand nucleic acid analysis. In particular, the invention relates tomethods, composition and apparatus for electron-induced fluorescent DNAsequencing.

[0003] 2. Background

[0004] With the advent of the human genome project, a need developed forimproved methods of sequencing nucleic acids such as DNA and RNA.Genetic information is stored in the form of very long molecules ofdeoxyribonucleic acid (DNA), organized into chromosomes. Thetwenty-three pairs of chromosomes in the human genome containapproximately three billion bases of DNA sequence. This DNA sequenceinformation determines multiple characteristics of each individual, suchas height, eye color and ethnicity. Many common diseases, such ascancer, cystic fibrosis, sickle cell anemia and muscular dystrophy arebased at least in part on variations in DNA sequence.

[0005] Determination of the entire sequence of the human genome hasprovided a foundation for identifying the genetic basis of suchdiseases. However, a great deal of work remains to be done to identifythe genetic variations associated with each disease. This requires DNAsequencing of portions of chromosomes in individuals or familiesexhibiting each such disease, in order to identify specific changes inDNA sequence that promote the disease. Ribonucleic acid (RNA), anintermediary molecule required for processing of genetic information,can also be sequenced to identify the genetic basis of various diseases.

[0006] Existing methods for nucleic acid sequencing, based on detectionof fluorescently labeled nucleic acids that have been separated by size,are limited by the length of the nucleic acid that can be sequenced.Typically, only 500 to 1,000 bases of nucleic acid sequence can bedetermined at one time. This is much shorter than the length of thefunctional unit of DNA, referred to as a gene, which can be tens or evenhundreds of thousands of bases in length. Determination of a completegene sequence requires that many copies of the gene be produced, cutinto overlapping fragments and sequenced, after which the overlappingDNA sequences can be assembled into the complete gene. This process islaborious, expensive, inefficient and time-consuming.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0008]FIG. 1 illustrates an embodiment of a microfluidic device.

[0009]FIG. 2 illustrates a cross-sectional view along the line 2-2 inFIG. 1.

[0010]FIG. 3 illustrates an embodiment of a detection unit.

[0011]FIG. 4 is a flow chart illustrating an embodied method.

[0012]FIG. 5 illustrates one embodiment of a micro column electron beamsource.

[0013]FIG. 6 illustrates one embodiment of an array of microfluidicsequencing units.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0014] Definitions

[0015] As used herein, “a” or “an” may mean one or more than one of anitem.

[0016] As used herein, the term “about” when applied to a number meanswithin plus or minus three percent of that number. For example, “about100” means any integer between 97 and 103.

[0017] “Nucleic acid” encompasses DNA, RNA, single-stranded,double-stranded or triple stranded and any chemical modificationsthereof, although single-stranded nucleic acids are preferred. Virtuallyany modification of the nucleic acid is contemplated by this invention.

[0018] Within the practice of the present invention, a “nucleic acid”may be of almost any length, from 10, 20, 50, 100, 200, 300, 500, 750,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000,9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,150,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000 or even morebases in length, up to a full-length chromosomal DNA molecule.

[0019] The apparatus, compositions and methods described herein can beused to sequence large complex genomes. Advantageously, intensive manualprocedures involving size fractionation of nucleic acid fragments onpolyacrylamide gels are avoided. The apparatus, compositions and methodscan provide high speed, small quantity, long read length nucleic acidsequencing. Information about a biological agent or a patient may beobtained in a timely and cost effective manner. The information obtainedfrom the nucleic acid sequence may be used to determine and initiateeffective countermeasures, such as vaccine administration, anti-viraladministration, patient monitoring, or treatment.

[0020] In certain embodiments a small quantity of a target nucleic acidto be sequenced may be hybridized with a probe library or a plurality ofprobe libraries. Probe libraries can be a group of oligonucleotides oroligonucleotide analogs. In preferred embodiments, each probe in alibrary is uniquely and detectably labeled. By identifying the probeshybridized to a target nucleic acid, the linear order of probes can beanalyzed and a nucleic acid sequence determined for the target. Incertain embodiments, analysis of a target nucleic acid molecule(s)hybridized to a series of probes can entail monitoring the targetnucleic acid using a detection unit operatively connected to amicrofluidic apparatus. In particular embodiments one or moremicrofluidic chambers, channels, capillaries, pores, valves orcombinations thereof may be used to process, move, and/or position atarget nucleic acid for analysis. A microfluidic device may be designedto position a nucleic acid molecule(s) in an extended conformation foranalysis. In certain embodiments, the target nucleic acid may bemanipulated so that only a single target nucleic acid molecule movesalong a microchannel at a time.

[0021]FIG. 1 illustrates an exemplary embodiment of an apparatus thatmay be used in the practice of certain methods of the invention. Adetection unit 1 may be positioned to monitor target nucleic acidmolecules flowing through a channel 2. The apparatus will typicallyinclude an input or reaction chamber 3 where one or more target nucleicacids may be hybridized to a probe library. An input chamber 3 may havean inlet port 4 and an outlet port 5 to provide for the flow ofreagents. Flow in and out of input chamber 3 can be controlled bymicrovalves 6 and the like operatively connected to the inlet port 4 andoutlet port 5.

[0022] A focusing region 7 may be used to hydrodynamically focus fluidscontaining hybridized target nucleic acid(s). Hydrodynamic focusing mayseparate one or more target nucleic acid molecules as fluid flows froman input chamber 3 to a channel 2. As a target molecule moves through afocusing region 7 it is extended to an approximate linear conformation.

[0023] A channel 2 may be positioned to flow fluid or solutions by adetection unit 1. A detection unit 1 will typically detect the spectralsignature of each labeled probe, preferably in sequential order. Adetection unit 1 may be operatively connected to a data processingsystem 8 for storage and analysis of detected spectra.

[0024] The channel 2 will have a lumen or groove that is in fluidcommunication with a fluid focusing region and an output chamber 9. Anoutput chamber 9 may be used for collecting or sorting fluids flowingout of channel 2. An output chamber 9 may also be operatively connectedto means for producing a motive force for fluid flow. Means forproducing a motive force include but are not limited to thermal,electroosmotic, pressure, and/or vacuum gradients.

[0025]FIG. 2 illustrates a cross-sectional view of the apparatus of FIG.1 along the line 2-2. The illustration in FIG. 2 shows an input chamber3 in fluid communication with a channel 2. The channel 2 is in fluidcommunication with an output chamber 9. The channel 2 may be positionedadjacent to a detector 20 which is part of the detection unit 1.Included in the detection unit 1 can be an excitation source 21,preferably a micro electron beam column. In certain embodiments thedetector may be operatively connected to a data processing system. Alsoillustrated in the cross-sectional view are fluid control means 22. Thefluid control means 22 may be sources of pressure, vacuum, heat,cooling, electric potential or other gradients to control the flow offluids within the apparatus. The excitation source 21 may be positionedto provide excitation energy to labeled probes as they flow in thechannel 2. Energy from the excitation source 21 may be absorbed by alabeled probe molecule and portions of that energy may be emitted fromthe label as a detectable signal, preferably a label specific spectralsignal.

[0026]FIG. 3 illustrates an expanded view of a signal-monitoring portionof an exemplary apparatus. In one embodiment, one or more probes 30 areassociated with the target nucleic acid molecule(s) 31 in a sequencespecific manner, preferably by Watson-Crick base pairing, forming adouble-stranded hybridized nucleic acid 33. In certain embodiments oneor more labels 32 uniquely identify each probe 30. A probe may bedetected as it moves past detection unit 34. In preferred embodiments,individual probes 30 are detected in sequential order as they move pastdetection unit 34 in a linear sequence, corresponding to the linearsequence of the target nucleic acid 31.

[0027] Detection unit 34 is generally comprised of an excitation source35 and a detector 36. In one embodiment the excitation source 35comprises a micro electron beam device or a micro e-beam 37. The microe-beam 37 may be focused to pass through a window 39 in the channel 40and within an appropriate distance of a probe 30 in order to excite theprobe 30 and transfer energy to a label 32 attached to the probe 30 bycoulombic induction. An excited label 32 subsequently emits a signal 38that is monitored by a detector 36. Preferably the signal 38 is anemission spectrum unique to the particular label 32. In certainembodiments, the time of each probe 30 passing a detector 36 isdetermined and recorded. Thus, if a hybridized nucleic acid 33 ispassing a detector 36 at a constant speed (nucleotide per time unit),gaps in a probe hybridization pattern can be detected. Such gaps mayoccur where hybridization between the target nucleic acid 31 and thelibrary of probes 30 is incomplete. Hybridization gaps can be addressedby using multiple hybridizations with multiple pools of probes 30 or byanalyzing a plurality of target nucleic acid molecules 33 that have beenhybridized to one or more probe libraries.

[0028]FIG. 4 is a flow chart representing an exemplary method. Block 200represents the isolation or immobilization of a target nucleic acid thatis a candidate for sequencing. Block 201 represents the synthesis of aprobe library, including the attachment of a unique label to each probeof the library. Block 202 represents the mixing and hybridization of atarget nucleic acid solution and a probe library, preferably within aninput chamber of a microfluidic device. Block 203 represents the movingof a hybridized nucleic acid in a channel past a detection unit. Block204 represents the detection of signals produced by labels attached toprobes that are hybridized to a target nucleic acid. An excitationsource, preferably an electron beam, is used to excite a label resultingin the emission of a signal as the label returns to its ground state.The analysis of the signals detected is represented in block 205. Thesignals that are detected by a detector can be analyzed by a dataprocessing system. Analysis typically entails the compilation of datasets, the determination of temporal occurrence of each spectrum, and anassignment of probe sequence to each spectrum resulting in a linearnucleic acid sequence.

[0029] In certain embodiments one or more hybridized target nucleic acidmolecules can be analyzed sequentially, concurrently or in parallel. Inone embodiment hybridized target nucleic acids may be passed by thedetection unit sequentially, that is, one nucleic acid at a time. Inanother embodiment a plurality of nucleic acids may be passed by adetection unit concurrently, that is, more than one at a time. In stillother embodiments hybridized target nucleic acid may be analyzedsequentially or concurrently in parallel processing using an array ofmicrofluidic devices fabricated on a single surface. The data obtainedcan be converted into a nucleic acid sequence by statistical analysisand processing of the signals detected by a data processing system.

[0030] Following detection and analysis by the described methods, onemay compare the nucleic acid sequence determined for a given sample orpatient with a statistically significant reference group of organisms ornormal patients and patients exhibiting a disease. In this way, it ispossible to correlate characteristics of the nucleic acids with variousorganisms or clinical states. The skilled artisan will understand thatrandom sequencing of the total genomic DNA of a sample or individual isnot a particularly efficient way in which to identify or detect DNAmarkers for a disease state or physiological condition. Typically,mapping or linkage studies are performed, in some cases in a family ofrelated individuals exhibiting a high occurrence of a disease state orcondition, in order to identify a portion of a chromosome associatedwith the state or condition. That portion of chromosomal DNA may betargeted for analysis by the sequencing methods of the presentinvention, preferably by isolating the nucleic acid segment of interestprior to sequencing.

[0031] Target Nucleic Acids

[0032] Nucleic acid molecules to be sequenced (target nucleic acids) maybe prepared by any standard technique. In one embodiment, the nucleicacids may be naturally occurring DNA or RNA molecules. Virtually anynaturally occurring nucleic acid may be prepared and sequenced by themethods of the present invention including, without limit, chromosomal,mitochondrial or chloroplast DNA or messenger, heterogeneous nuclear,ribosomal or transfer RNA.

[0033] Methods for preparing and isolating various forms of cellularnucleic acids are known. In general cells, tissues or other sourcematerial containing nucleic acids to be sequenced must first behomogenized, for example by freezing in liquid nitrogen followed bygrinding in a morter and pestle. Certain tissues may be mechanicallyhomogenized using a Waring blender, Virtis homogenizer, Douncehomogenizer or other homogenizer. Crude homogenates may be extractedwith detergents, such as sodium dodecyl sulphate (SDS), Triton X-100,CHAPS, octylglucoside or other detergents known in the art.Alternatively or in addition, extraction may use chaotrophic agents suchas guanidinium isothiocyanate, or organic solvents such as phenol. Insome embodiments, protease treatment, for example with proteinase K, maybe used to degrade cell proteins. Particulate contaminants may beremoved by centrifugation. Dialysis against aqueous buffer of low ionicstrength may be of use to remove salts or other soluble contaminants.Nucleic acids may be precipitated by addition of ethanol at −20° C., orby addition of sodium acetate (pH 6.5) to a final concentration of 0.3 Mand 0.8 volumes of 2-propanol. Precipitated nucleic acids may becollected by centrifugation or, for chromosomal DNA, by spooling theprecipitated DNA with a glass pipet.

[0034] The procedures listed above are exemplary only and that manyvariations may be used within the scope of the present invention,depending on the particular type of target nucleic acid. For example,mitochondrial DNA is often prepared by cesium chloride density gradientcentrifugation, using step gradients, while mRNA is often prepared usingpreparative columns from commercial sources, such as Promega (Madison,Wis.) or Clontech (Palo Alto, Calif.).

[0035] Depending on the type of nucleic acid to be prepared variousnuclease inhibitors may be required. For example, RNase contamination inbulk solutions may be eliminated by treatment with diethyl pyrocarbonate(DEPC), while commercially available nuclease inhibitors may be obtainedfrom standard sources such as Promega (Madison, Wis.) or BRL(Gaithersburg, Md.). Purified nucleic acid may be dissolved in aqueousbuffer, such as TE (Tris-EDTA) and stored at −20° C. or in liquidnitrogen prior to use.

[0036] In cases where single stranded DNA (ssDNA) is to be sequenced, anssDNA may be prepared from double stranded DNA (dsDNA) by standardmethods. Most simply, dsDNA may be heated above its annealingtemperature, at which point it spontaneously disaggregates into ssDNA.Representative conditions might involve heating at 92 to 95° C. for 5min or longer.

[0037] Although the discussion above concerns preparation of naturallyoccurring nucleic acids virtually any type of nucleic acid that is ableto hybridize with a probe library of the invention could potentially besequenced by the methods of the present invention. For example, nucleicacids prepared by various standard amplification techniques, such aspolymerase chain reaction (PCR™) amplification, could be sequencedwithin the scope of the present invention.

[0038] Target nucleic acid templates may be isolated from a wide varietyof organisms including, but not limited to, viruses, bacteria,eukaryotes, mammals, and humans. In certain embodiments target nucleicacid(s) that are isolated and amplified via cloning into plasmids, M13,lambda phage, P1 artificial chromosomes (PACs), bacterial artificialchromosomes (BACs), yeast artificial chromosomes (YACs) and othercloning vectors may be sequenced using the methods of the invention.Also contemplated for use are amplified nucleic acids or amplifiedportions of nucleic acids. In specific embodiments a cloning vector canbe linearized, immobilized, denatured and hybridized with a set(s) ofprobe libraries.

[0039] Nucleic Acid Amplification

[0040] Nucleic acids that may be used as templates for amplification aretypically isolated from cells contained in biological samples accordingto standard methodologies. The nucleic acid may be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary cDNA. In one embodiment, the RNA iswhole cell RNA and is used directly as the template for amplification.

[0041] A number of template dependent processes are available to amplifytarget nucleic acid sequences present in a given sample. One of thebest-known amplification methods is the polymerase chain reaction (PCR).Another method for nucleic acid amplification is the ligase chainreaction (“LCR”). In yet another method of nucleic acid amplificationQbeta Replicase may also be used. Strand Displacement Amplification(SDA) is another method of carrying out isothermal amplification ofnucleic acids that involves multiple rounds of strand displacement andsynthesis, i.e., nick translation. Other nucleic acid amplificationprocedures include transcription-based amplification systems (TAS),including nucleic acid sequence based amplification (NASBA) and 3SR.

[0042] Probe Libraries

[0043] The term “probe” denotes a defined nucleic acid segment such asDNA or RNA, or any analog thereof, such as peptide nucleic acid (PNA),which can be used to identify a specific complementary nucleic acidsequence in a target nucleic acid.

[0044] In certain embodiments one or more probe libraries may beprepared for hybridization to one or more target nucleic acid molecules.For example, a set of relatively short probes containing all 4096 orabout 2000 non-complementary 6-mers, or all 16,384 or about 8,000non-complementary 7-mers may be used. A set or plurality of subsets of8-mers and longer probes may be used that include 65,536 or about 32,000non-complementary 8-mers. If non-complementary subsets of probes are tobe used a plurality of hybridizations and sequence analyses may becarried out and the results of the analyses merged into a single dataset by computational methods. For example, if a library comprising onlynon-complementary 6-mers were used for hybridization and sequenceanalysis, a second hybridization and analysis using the same targetnucleic acid molecule hybridized to those probe sequences excluded fromthe first library would be performed.

[0045] In certain preferred embodiments, the probe library contains allpossible nucleic acid sequences for a given probe length (e.g., asix-mer library would consist of 4096 probes). In such cases, certainprobes will form hybrids with complementary probe sequences. Suchprobe-probe hybrids, as well as unhybridized probes, may be excludedfrom the channel leading to the detection unit.

[0046] Methods for the selection and generation of complete sets orspecific subsets of probes of all possible sequences for a given probelength are known. In general, for a probe of length “n”, a complete setof all possible probe sequences can be represented as 4^(n) probes. Asthe probe length increases so does the number of distinct label moietiesneeded to uniquely label each probe. Limiting the number of labelmolecules required would involve the use of the shortest possible probelength consistent with the requirements for stable probe hybridizationto the target molecule. In various embodiments, probes of 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides in length may beused within the scope of the present invention. The length of a probe(s)will vary with hybridization, wash conditions and composition of probelibrary or libraries used. Generally, probes may range from about 2 toabout 20 monomers in length, with from about 4 to about 15 beingpreferred and from about 6 to about 10 being more preferred.

[0047] In certain embodiments, it may be useful to use probes comprisinga random nucleic acid sequence in the middle of the probe and a constantnucleic acid sequences at one or both ends, a random portion andconstant portion, respectively. This may be of use, for example, wherethe number of distinct label moieties available is lower than the numberof possible probe sequences required for a probe length that is optimalfor hybridization. For example, a subset of 12-mer probes could consistof a complete set of random 8-mer sequences attached to constant 2-mersat each end. Alternatively, a subset of 10-mer probes could consist of acomplete set of random 8-mers attached to constant 1-mers at each end,or constant 2-mers at one end. These probe libraries can be subdividedaccording to their constant portions and hybridized separately to atarget nucleic acid followed by analysis using the combined data of eachdifferent probe library to determine the nucleic acid sequence. Theskilled artisan will realize that the number of sublibraries requiredwill be a function of the number of constant bases that are attached tothe random sequences. For example, with one constant base attached tothe random sequences a total of four sublibraries might be hybridized tothe target. For two constant bases attached to the random sequences, atotal of sixteen sublibraries might be hybridized to the target, one foreach possible contant region.

[0048] An alternative embodiment may use multiple hybridizations andanalyses with a single probe library containing a specific constantportion attached to random probe sequences. For any given site on atarget nucleic acid, it is possible that multiple probes of different,but overlapping sequence could bind to that site in a slightly offsetmanner. Thus, using multiple hybridizations and analyses with a singlelibrary, a complete sequence of the target nucleic acid could beobtained by compiling the overlapping, offset probe sequences. Althoughthis approach is somewhat similar to the “shotgun” methods used withstandard sequencing, it avoids having to generate and clone multiplerestricted fragments for sequencing, since only a single target nucleicacid sequence is required. Random nucleic acid sequences of a probe mayinclude, but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore bases.

[0049] Each probe may have at least one covalently attached label ortag. In certain embodiments the probes may have a plurality of attachedlabels or tags, the combination of which is unique to a particularprobe. Combinations of labels can be used to expand the number of uniquelabels available for specifically identifying a probe in a library. Inother embodiments the probes may each have a single unique labelattached. Probes with a single label as well as probes with a pluralityof labels are contemplated. The only requirement is that the signaldetected from each probe must be capable of uniquely identifying thesequence of that probe.

[0050] 1. Oligonucleotide Libraries

[0051] In certain embodiments of the invention the probe library is anoligonucleotide probe library. Oligonucleotide probes may be prepared bystandard synthetic methods. Alternatively, probes can be purchased froma variety of vendors or synthesized on instrumentation such as theApplied Biosystems 381A DNA synthesizer (Foster City, Calif.) or similarinstruments. In certain embodiments, probes may be labeled withfluorescent dyes or other types of labels. In preferred embodimentsconductive polymers may be used to label oligonucleotide probes. Inembodiments where probes are chemically synthesized, the label moietymay be covalently attached to one or more of the nucleotide precursorsused for synthesis. Alternatively, the label may be attached after theprobe has been synthesized.

[0052] In various embodiments, the sequence of the probe may be knownbefore the label is attached, or the probe sequence attached to a givenlabel may be identified after attachment. For example, a nucleic acidchip containing sequences complementary to all possible probe sequencesmay be designed, each sequence assigned to a given location on the chip.After hybridization to a library of labeled probes, the emissionspectrum of the label at each position on the chip may be identified.

[0053] 2. Peptide Nucleic Acids (PNAs)

[0054] In alternative embodiments peptide nucleic acids (PNAs) may beused as probes. PNAs are a polyamide type of DNA analog with monomericunits for adenine, guanine, thymine, and cytosine. PNAs are availablecommercially from companies such as Perceptive Biosystems. The backboneis made from repeating N-(2-aminoethyl)-glycine units linked by peptidebonds. The different bases (purines and pyrimidines) are linked to thebackbone by methylene carbonyl linkages. Unlike DNA or other DNAanalogs, PNAs do not contain any pentose sugar moieties or phosphategroups.

[0055] PNA synthesis may be performed with 9-fluoroenylmethoxycarbonyl(Fmoc) monomer activation and coupling usingO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) in the presence of a tertiary amine,N,N-diisopropylethylamine (DIEA). PNAs can be purified by reverse phasehigh performance liquid chromatography (RP-HPLC) and systematicallyverified by matrix assisted laser desorption ionization—time of flight(MALDI-TOF) mass spectrometry analysis.

[0056] Labeling of Probes

[0057] In certain embodiments one or more labels may be attached to eachprobe. The label will typically produce a detectable signal.Non-limiting examples of labels that could be used include fluorescent,luminescent, radioactive, phosphorescent, chemiluminescent, enzymatic,spin, electron dense, or mass spectroscopic labels. A label may bedetected by using a variety of means, such as a spectrophotometer,luminometer, NMR (nuclear magnetic resonance), mass-spectroscopy,imaging systems, photomultiplier tube and/or other appropriate standarddetection means. In certain embodiments conductive polymers may be usedas a label. Conductive polymers are tunable to unique spectroscopicprofiles based on the polymer composition, length, side chain groupsand/or dopants.

[0058] Labels may be coupled to amine or thiol groups incorporated intoa probe during synthesis. Labels can be coupled internally or to eitherterminus of a probe. A label(s) may be linked to a monomer unit of aprobe via a spacer arm to reduce steric hindrance with, for example,hydrogen bond formation during probe hybridization. After synthesis,probes may be purified by high performance liquid chromatography (HPLC)or polyacrylamide gel electrophoresis (PAGE).

[0059] In certain embodiments a label may be incorporated into aprecursor prior to the synthesis of a probe. Internalamino-modifications for labeling at “A” and “G” positions are alsocontemplated. Typically, when internal labeling is required, it isperformed at a “T” position using a commercially availablephosphoramidite. In certain embodiments library segments with apropylamine linker at the A and G positions are used to internally labela probe. The introduction of an internal aminoalkyl tail allowspost-synthetic labeling of the probe. Linkers may be purchased fromvendors such as Synthetic Genetics, San Diego, Calif.

[0060] In one embodiment automatic coupling using the appropriatephosphoramidite derivative of the label is also contemplated. Theselabels are coupled during the synthesis at the 5′-terminus.

[0061] 1. Conductive Polymers

[0062] In certain embodiments conductive polymers may be used as alabel. Conductive polymers are long, carbon-based chains composed ofsimple repeating units. Conductive polymers are generally formed byconverting single-bond carbon chains to polymer backbones withalternating single and double bonds. This change typically provides apathway for free-electron-charge carriers. Typical conductive-polymersinclude, but are not limited to polyaniline, polyphenylene-vinylene,polythiophene, polypyrrole, polyacetylene, polydiacetylene,polytriacetylene, poly-p-phenylene, polyfuran, betacarotene, substitutedforms of these molecules and other similar conjugated oligomermaterials. Conductive polymers may be composed of the same repeatingunit (conductive polymer) or alternately different repeating units(conductive oligomer).

[0063] A. Tuning of Conductive Polymers

[0064] Optical transitions of conductive polymers may be tuned tocertain emission spectra by the length and/or the composition of sidechains of the conductive polymer. A non-limiting example is thealteration in the emission spectra of polyphenylene-vinylene (PPV) byalteration of side chains (R groups). Substitution of different sidechains onto PPV, such as alkoxy groups will result in shift of theemission spectra to the red portion of the light spectrum. Substitutionof an alkyl or an aryl side chain will lead to an emission shift to theblue end of the light spectrum.

[0065] By adjusting the length and/or side chain composition, the numberof distinguishable conductive polymer labels may be veryhigh—potentially in the tens of thousands or higher. Thus, conductivepolymers may be of use in embodiments requiring large numbers ofdistinguishable label moieties.

[0066] B. Attachment of Conductive Polymers

[0067] Conductive polymers may be covalently attached to a probe. Theprobes may be oligonucleotides, PNAs or analogs thereof that hybridizesequence specifically to target nucleic acids. In general, the covalentattachments are made in such a manner as to minimize steric hindrance ofa probe and any adjacent labels that may be attached to the same probeor adjacent probes. Linkers may be used that are generally short andprovide a degree of flexibility to the label and labeled probe. In acertain embodiments the techniques outlined above may be used to attacha conductive polymer to nucleic acids and nucleic acid analogs,including PNAs.

[0068] The point of attachment to a base will vary with the base. Whileattachment at any position is possible, it is preferred to attach atpositions not involved in hydrogen bonding to the complementary base.Thus, for example, attachment can be to the 5 or 6 positions ofpyrimidines such as uridine, cytosine and thymine. For purines such asadenine and guanine, the linkage is preferably via the 8 position.Attachment to nonstandard bases is preferably done at the comparablepositions.

[0069] In one embodiment, conductive polymers with terminal acetylenebonds may be attached directly to the base. Homo-or hetero-bifunctionallinkers are available from various commercial sources. Linkers include,but are not limited to, alkyl groups and alkyl groups containingheteroatom moieties, with short alkyl groups, esters, epoxy groups andethylene glycol and derivatives being preferred. Propyl, acetylene, andC2alkene groups are especially preferred. Linkers may also comprise asulfone group, forming sulfonamide linkages. In particular embodiments,nucleosides may be modified with amino groups, sulfur groups, siliconegroups, phosphorus groups, or oxo groups. These modified nucleosides arethen used to attach the conductive polymers.

[0070] 2. Fluorescent Labels

[0071] In certain embodiments fluorescent dyes may be used to labelprobes. Fluorophores include but are not limited to (Fluorophore(Excitation/Emission))—FAM (488 nm/518 nm); HEX (488 nm/556 nm); TET(488 nm/538 nm); CY (3550 nm/570 nm); CY5 (649 nm/670 nm); CY5.5 (675nm/694 nm); JOE (527 nm/548 nm); 6-ROX (575 nm/602 nm); Cascadde Blue(400 nm/425 nm); Texas Red (595 nm/615 nm); Rhodamine (550 nm/575 nm);Rhodamine green (502 nm/527 nm); Rhodamine red (570 nm/590 nm);Rhodamine 6G (525 nm/555 nm); 6-TAMRA (555 nm/580 nm); 5-TAMRA (543nm/567 nm); Alexa 430 (430 nm/545 nm); Alexa 488 (493 nm/516 nm); Alexa594 (588 nm/612 nm); Bodipy R6G (528 nm/550 nm). These fluorescentlabels are commercially available from a variety of vendors. Additionalfluorescent labels include Tetramethyl rhodamine (argon laser),fluorscein, 9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene and itsderivatives, 7-nitrobenzofurazan, and NBD. Xanthene dyes may becovalently attached through the carboxylic acid functionality, via anamide bond with a linker amine group. Fluorescent dyes may be used inconjunction with each other and/or quencher molecules to expand thenumber of spectrally unique labels. Other alternative labels or tagsinclude semiconductor nanostructures or quantum dots that may becovalently attached to a probe.

[0072] Hybridization

[0073] Hybridization includes, but is not limited to forming a doublestranded molecule or forming a molecule with a partial double strandednature. In preferred embodiments, hybridization of the target nucleicacid to the probe library occurs under stringent conditions that allowhybridization between fully complementary nucleic acid sequences, butpreclude hybridization between partially mis-matched sequences. Forexample, hybridization at low temperature and/or high ionic strength istermed low stringency. Hybridization at high temperature and/or lowionic strength is termed high stringency. Low stringency is generallyperformed at 0.15 M to 0.9 M NaCl at a temperature range of 20° C. to50° C. High stringency is generally performed at 0.02 M to 0.15 M NaClat a temperature range of 50° C. to 70° C.

[0074] It is understood that the temperature and/or ionic strength of adesired stringency are determined in part by the length of theparticular probe, the length and/or base content of the targetsequences, and the presence of formamide, tetrametylammonium chloride orother solvents in the hybridization mixture. Various standardhybridization solutions are well known. It is also understood that theseranges are mentioned by way of example only and that the desiredstringency for a particular hybridization reaction is often determinedempirically by comparison to positive and/or negative controls.Accordingly, the probes of the disclosure may be used for their abilityto selectively form duplex molecules by hybridization with complementarystretches of target nucleic acids, such as DNA or RNA.

[0075] It is preferred to employ relatively stringent conditions to formhybridized nucleic acids comprising a target nucleic acid and one ormore probe molecules. For example, relatively low salt and hightemperature conditions, such as provided by about 0.02 M to about 0.10 MNaCl at temperatures of about 50° C. to about 70° C. Such highstringency conditions tolerate little, if any, mismatch between theprobe and the target strand. It is generally appreciated that conditionsmay be rendered more stringent by the addition of increasing amounts offormamide or the like.

[0076] Processing of Hybridized Nucleic Acid Template

[0077] Once a target nucleic acid is hybridized to a probe library thehybridized nucleic acid will typically be processed. In certainembodiments non-hybridizing probes may be separated from a hybridizednucleic acid. A hybridized nucleic acid may be separated fromnon-hybridized probes before or after introduction into a microfluidicdevice. The separation of non-hybridized probes may be accomplished by,for example, selective precipitation, size exclusion columns, selectiveimmobilization of a target nucleic acid or any equivalent technique.

[0078] A target nucleic acid may be immobilized in an input chamber of amicrofluidic device by optical trapping, magnetic trapping, attachmentto a chamber wall and similar immobilization methods. A target nucleicacid may be attached to an input chamber wall and subsequentlyhybridized to probe libraries. After incubation with an immobilizedtarget nucleic acid non-hybridized probes can be washed out of an inputchamber using inlet and outlet ports in a microfluidic device. Onceseparated from non-hybridized probes, a hybridized nucleic acid can bereleased from an input chamber wall and manipulated through amicrochannel, microcapillary, or micropore for analysis. Alternatively,a hybridized nucleic acid that has been separated from non-hybridizedprobes by selective precipitation, size exclusion columns, or the likemay be introduced into an input chamber and manipulated through amicrochannel, microcapillary, or micropore for analysis.

[0079] Once non-hybridized probes are separated, a hybridized nucleicacid may be further manipulated by microfluidic sorting, fluidicfocusing, or combinations thereof. Hydrodynamics on a micron scale canbe used to manipulate the flow of nucleic acids into a microchannel,microcapillary, or a micropore and past a detector where the linearorder of hybridized probes can be determined. In one embodiment of theinvention microfluidics is used to sort hybridized nucleic acid(s).Microfluidic sorting of hybridized nucleic acids may include filteringof a solution containing hybridized nucleic acids across a comb toseparate individual molecules which are subsequently moved into amicrochannel, microcapillary, or a micropore one or more molecule at atime. Manipulation of the fluid flow may be done by using electric,thermal, pressure or vacuum motive forces.

[0080] In another embodiment microfluidics is used to fluidically focushybridized nucleic acid(s). Fluidic focusing of hybridized nucleic acidsmay include moving a fluid containing hybridized nucleic acids through anarrowing path at an appropriate velocity to produce a focused stream offluid flow, thus separating individual molecules and moving themolecules into a microchannel, microcapillary, or a micropore one ormore molecule at a time. Manipulation of the fluid flow may be done byusing electric, thermal, pressure or vacuum motive forces.

[0081] 1. Microfluidics

[0082] Microfluidics is defined as the domain of fluidics where thefluids have a Reynolds number much smaller than one, which implies thatthe fluid flow is laminar. A Reynolds number (Re) is a dimensionlessnumber used in fluid dynamics to describe the nature of fluid flow in aparticular situation: Reynolds number=density×flowvelocity×characteristic dimension/viscosity. At low Re the viscousforces dominate and extend over large distances causing laminar flow. Inmost practical cases, these low Reynolds numbers require the channels inwhich the fluid flows to measure in the tens of micrometers range orless.

[0083] The small dimensions also require precise dimensional control,since the fluidic properties are very sensitive to dimensionalvariations. When precise dimensional control is required, silicon is anexcellent material for construction. Photolithography techniques used inintegrated circuit processing have been used with this material toobtain channels in the range of tens of micrometers wide and deep.

[0084] The microfluidic devices of certain embodiments comprise acentral body structure in which the various microfluidic elements aredisposed. The body structure includes an exterior portion or surface, aswell as an interior portion that defines the various microscale channelsand/or chambers of the overall microfluidic device. For example, thebody structures of a microfluidic device typically employ a solid orsemisolid substrate that is typically planar in structure, i.e.,substantially flat or having at least one flat surface. Often, theplanar substrates are manufactured using solid substrates common in thefields of microfabrication, e.g., silica-based substrates, such asglass, quartz, silicon or polysilicon, as well as other knownsubstrates, i.e., gallium arsenide. In the case of these substrates,common microfabrication techniques, such as photolithographictechniques, wet chemical etching, micromachining, i.e., drilling,milling and the like, may be readily applied in the fabrication ofmicrofluidic devices and substrates. Alternatively, polymeric substratematerials may be used to fabricate the devices of the present invention,including, e.g., polydimethylsiloxanes (PDMS), polymethylmethacrylate(PMMA), polyurethane, polyvinylchloride (PVC), polystyrene polysulfone,polycarbonate, polymethylpentene, polypropylene, polyethylene,polyvinylidine fluoride, ABS (acrylonitrile-butadiene-styrenecopolymer), and the like. In the case of such polymeric materials,injection molding or embossing methods may be used to form thesubstrates having the channel and reservoir geometries as describedherein.

[0085] The channels and chambers of the device are typically fabricatedinto one surface of a planar substrate, as grooves, wells or depressionsin that surface. A second planar substrate, typically prepared from thesame or similar material, is overlaid and bonded to the first, therebydefining and sealing the channels and/or chambers of the device.Together, the upper surface of the first substrate, and the lower matedsurface of the upper substrate define the channels and chambers of thedevice.

[0086] Movement of fluids through the device will typically be regulatedby computer-controlled pressure and electrokinetic forces to gain exactcontrol over the flow of fluids in microfluidic channels. Typically,electricity is used to drive samples through the channels. In one formof stimulation, electro-osmosis, computer-driven power supplies locatedin reservoirs at each end of a channel are activated to generateelectrical current through the channel. The current forces moleculeswith different electrical charges in fluids to travel through thechannels at different rates; a typical speed is one millimeter persecond. Another type of electrokinesis—electrophoresis—is simply amicrominiaturized version of an analytical approach routinely used inchemistry laboratories. An electric field influences the movement ofcharged molecules in microfluids moving through the channels. Electricfields can be used to move molecules in solution or to separatemolecules with very subtle differences.

[0087] Alternate methods for moving microfluids include temperaturegradients or micropressure. For example, small amounts of pressureapplied to microfluids traveling through channels create predictable andreproducible flows. Temperature gradients can move tiny volumes ofliquids around nano-sized canals in silicon wafers. This occurs becausethe surface tension of the microfluids varies with temperature; atemperature gradient of just three or four degrees Celsius is enough tocause a microfluid to seek a cold region in its pathway. Chemicalmodifications to the substrate applied by lithography amplify the effectby creating a series of chemical levees along the canals.

[0088] Optical Trapping

[0089] In alternative embodiments an optical trap can be used tomanipulate a target nucleic acids. An optical trap is a device in whicha particle can be trapped near the focus of a strongly focused lightbeam such as a laser beam. The particle is held in the trap by the axialgradient force that is proportional to the gradient of the lightintensity and points in the direction of increased intensity. Ingeneral, single-beam optical trapping can be achieved for particleshaving sizes ranging from about 10 μm to about 10 nm. Commercial opticaltrapping systems are available, such as, LaserTweezers(tm) 2000, CellRobotics, inc.; Compact Photonic Tweezers, S+L Gmb; and PALM(tm)Laser-Microscope System, P.A.L.M. GmbH. In certain embodiments a targetnucleic acid may be immobilized onto a bead. The bead can then beoptically trapped within a hybridization chamber of a microfluidicdevice. Alternatively, a target nucleic acid may be immobilized onto amagnetic bead that is subsequently trapped magnetically.

[0090] Methods of Nucleic Acid Immobilization

[0091] In various embodiments, the nucleic acid molecules of the presentinvention may be attached or immobilized to a solid surface.Immobilization of nucleic acid molecules may be achieved by a variety ofmethods involving either non-covalent or covalent attachment between thenucleic acid molecule and a support or surface. In an exemplaryembodiment, immobilization may be achieved by coating a solid surfacewith streptavidin or avidin and the subsequent attachment of abiotinylated polynucleotide. Immobilization may also occur by coating apolystyrene, glass or other solid surface with poly-L-Lys or poly L-Lys,Phe, followed by covalent attachment of either amino- orsulfhydryl-modified nucleic acids using bifunctional crosslinkingreagents. Amine residues may be introduced onto a surface through theuse of aminosilane.

[0092] Immobilization may take place by direct covalent attachment of5′-phosphorylated nucleic acids to chemically modified polystyrenesurfaces. The covalent bond between the nucleic acid and the solidsurface is formed by condensation with a water-soluble carbodiimide.This method facilitates a predominantly 5′-attachment of the nucleicacids via their 5′-phosphates.

[0093] DNA is commonly bound to glass by first silanizing the glasssurface, then activating with carbodiimide or glutaraldehyde.Alternative procedures may use reagents such as3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane(APTS) with DNA linked via amino linkers incorporated either at the 3′or 5′ end of the molecule during DNA synthesis. DNA may be bounddirectly to membranes using ultraviolet radiation. Other non-limitingexamples of immobilization techniques for nucleic acids are known.

[0094] The type of surface to be used for immobilization of the nucleicacid is not limited to those disclosed above. In various embodiments,the immobilization surface could be magnetic beads, non-magnetic beads,a planar surface, a pointed surface, or any other conformation of solidsurface comprising almost any material, so long as the material willallow hybridization of target nucleic acids to probe libraries.

[0095] Bifunctional cross-linking reagents may be of use in variousembodiments, such as attaching a nucleic acid molecule to a surface.Exemplary cross-linking reagents include glutaraldehyde (GAD),bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), andcarbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC).

[0096] In certain embodiments a capture oligonucleotide may be bound toa surface. The capture oligonucleotide will hybridize with a specificnucleic acid sequence of a target nucleic acid. Once a target nucleicacid is hybridized to a capture oligo it may be covalently attached by aligation reaction. A target nucleic acid may be released from thesurface by restriction enzyme digestion, elevated temperature, reducedsalt concentration, or a combination of these and similar methods.

[0097] Detection Unit

[0098] In particular embodiments a detection unit may comprise anexcitation source and a detector. An excitation source can promotealterations in a label that can result in a signal emission. Forexample, an electron beam may excite electrons of a label by coulombicinduction, which may result in a fluorescent emission as an excitedlabel returns to its ground state. Emitted energy can be detected by avariety of detectors including, but not limited tomicrospectrophotometers, photomultiplier tubes and avalanche detectors.In a particular embodiment a micro electron beam may be used as anexcitation source. Preferably, the energy absorbed will be emitted as adetectable signal and each label will have a unique spectral profile.

[0099] Excitation Source

[0100] In certain embodiments the excitation source can be an electronbeam. In alternate embodiments the excitation source may generate otherforms of electromagnetic radiation such as UV, visible or infraredlight. Alternate embodiments rely on detection and identification ofprobes released after transit through a microchannel, microcapillary, ormicropore by hybridization to arrays, mass spectroscopy or nuclearmagnetic resonance. Probes may be released from a target nucleic acidafter transit through a microfluidic device by enzymatic degradation ofa target nucleic acid or disruption of the sequence specific binding ofthe probe to the target nucleic acid, for example by heating. Releasedprobes can be further manipulated by flow through an extendedmicrofluidic device to an appropriate detector.

[0101] In certain embodiments the excitation source may be a microcolumn electron beam (micro e-beam) source as illustrated in FIG. 5. Theexcitation source illustrated in FIG. 5 may be made by usingmicrofabrication techniques known in the art and is based largely on themicromachining of silicon. The stacked parts of the device would beassembled in a vacuum using a wafer-to-wafer bonding approach. Anelectron source 300 is positioned at the opening of an evacuated column301. The opening of the evacuated column 301 has an associatedlow-voltage gate electrode 302. Perpendicular to the evacuated column'slong axis are aligned high-voltage acceleration, deflection, andfocusing electrodes 303. The evacuated column is typically sealed at thefar end by an electron transparent membrane 304 that is typicallyoperatively coupled to a microchannel. The electron transparent membrane304 is positioned so that an electron beam can be focused onto achannel. The channel provides a microfluidic path for the passage oftarget nucleic acids hybridized to labeled probes. The e-beam can bedirected so it is incident with passing target nucleic acid(s) andexcites an associated labeled probe. The excited label typically emitsspectra of light characteristic of the probe. Photons of light aredetected by a detector array and may be stored in a processing unit forfurther analysis.

[0102] In one embodiment an array of micro column electron beam sourcesmay be fabricated onto a support. FIG. 6 shows an array of microfluidicelectron induced fluorescence sequencing apparatus 400. Each of themicrofluidic devices comprises a micro column electron beam source 401,an inlet port 402, an outlet port 403, an input chamber 404, fluidicfocusing region 405, microchannel 406, an output chamber 407, inlet exitport 408 and an outlet exit port 409. The input chamber 404 is in fluidcommunication with the output chamber 407 via the microchannel 406. Asingle hybridization mixture can be introduced to and analyzed in eachdevice 400 of the array. Alternatively, distinct hybridization mixturescan be introduced to and analyzed in separate devices 400 of the array.

[0103] Signal Detection

[0104] Signals produced by one or more labeled probes may be detected byvarious detection means. In certain embodiments the energy released by alabel can be detected using sensitive detection means such as aspectrophotometer, photomultiplier tube, charge-coupled device, oravalanche photodiode. In particular embodiments light emitted by a labelcan be analyzed by a Jobin-Yvon HRD1 double grating monochromator anddetected by a GaAs cathode photo-multiplier tube, then fed to a computerthrough a photon counter. Alternatively, the detectors may be used inconjunction with fluorescent microscopy.

[0105] Determination of Nucleic Acid Sequence

[0106] In one embodiment a nucleic acid sequence may be determined byusing a decoding method. In certain embodiments, a decoding method, asillustrated by the flow chart in FIG. 4, may entail the immobilization asingle stranded DNA or a double stranded DNA 200 followed bydenaturation to provide a target single-stranded nucleic acid sequence.A probe library or a plurality of probe libraries may be created 201such that each probe of the library will have an associated label thatwill specifically and uniquely identify the probe. The target nucleicacid sequence is incubated with a probe library or series of probelibraries to allow hybridization of the probes to the target sequence202. After hybridization of the probes the hybridized nucleic acids aremanipulated through a micro-fluidic channel where they flow past anexcitation source and a detector 203. Emission spectra of the labeledprobes may then be detected and relayed to a data processing system 204.The sequence of the target nucleic acid is determined by comparing theemission spectra and the order in which the emission spectra weredetected to a database of spectra for labels associated with the probes205. The linear sequence of probes hybridized to the target nucleic acidcan then be determined by either statistical calculations or thedetermination of probe order from a single target nucleic acid molecule.

[0107] In alternate embodiments of the invention individual probeshybridized to the target nucleic acid may be covalently joined toadjacent probes by ligation. Ligation of probes would produce longersegments of hybridized sequences and a more stable association betweenprobe and target. The data collected may be analyzed and translated intothe target nucleic acid sequence by using a computer implemented systemdesigned for such analysis.

[0108] The length of nucleic acid analyzed by this method should only belimited by the length of nucleic acid that can be manipulated with outshearing, breaking or other means of degradation of a target nucleicacid, allowing for extended read lengths and faster, more economicalsequencing of nucleic acids.

[0109] Information Processing and Control System and Data Analysis

[0110] In certain embodiments, the sequencing apparatus may beinterfaced with an information processing and control system. In anexemplary embodiment, the system incorporates a computer comprising abus or other communication means for communicating information, and aprocessor or other processing means coupled with the bus for processinginformation. In one embodiment, the processor is selected from thePentium® family of processors, including the Pentium® II family, thePentium® III family and the Pentium® 4 family of processors availablefrom Intel Corp. (Santa Clara, Calif.). In alternative embodiments, theprocessor may be a Celeron®, an Itanium®, or a Pentium Xeon® processor(Intel Corp., Santa Clara, Calif.). In various other embodiments, theprocessor may be based on Intel® architecture, such as Intel® IA-32 orIntel® IA-64 architecture. Alternatively, other processors may be used.

[0111] The computer may further comprise a random access memory (RAM) orother dynamic storage device (main memory), coupled to the bus forstoring information and instructions to be executed by the processor.Main memory may also be used for storing temporary variables or otherintermediate information during execution of instructions by processor.The computer may also comprise a read only memory (ROM) and/or otherstatic storage device coupled to the bus for storing static informationand instructions for the processor. Other standard computer components,such as a display device, keyboard, mouse, modem, network card, or othercomponents known in the art may be incorporated into the informationprocessing and control system. The skilled artisan will appreciate thata differently equipped information processing and control system thanthe examples described herein may be desirable for certainimplementations. Therefore, the configuration of the system may varywithin the scope of the present invention.

[0112] In particular embodiments, the detection unit may also be coupledto the bus. Data from the detection unit may be processed by theprocessor and the processed and/or raw data stored in the main memory.Data on known emission spectra for labeled probes may also be stored inmain memory or in ROM. The processor may compare the emission spectrafrom probes in the channel to the stored spectra to identify thesequence of probes passing along the channel. The processor may analyzethe data from the detection unit to determine the sequence of the targetnucleic acid.

[0113] The information processing and control system may further provideautomated control of the sequencing apparatus. Instructions from theprocessor may be transmitted through the bus to various output devices,for example to control the pumps, electrophoretic or electro-osmoticleads and other components of the apparatus.

[0114] It should be noted that, while the processes described herein maybe performed under the control of a programmed processor, in alternativeembodiments, the processes may be fully or partially implemented by anyprogrammable or hardcoded logic, such as Field Programmable Gate Arrays(FPGAs), TTL logic, or Application Specific Integrated Circuits (ASICs),for example. Additionally, the method of the present invention may beperformed by any combination of programmed general purpose computercomponents and/or custom hardware components.

[0115] In certain embodiments, custom designed software packages may beused to analyze the data obtained from the detection unit. Inalternative embodiments, data analysis may be performed using aninformation processing and control system and publicly availablesoftware packages. Non-limiting examples of available software for DNAsequence analysis includes the PRISM™ DNA Sequencing Analysis Software(Applied Biosystems, Foster City, Calif.), the Sequencher™ package (GeneCodes, Ann Arbor, Mich.), and a variety of software packages availablethrough the National Biotechnology Information Facility at websitewww.nbif.org/links/1.4.1.php.

EXAMPLES

[0116] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Probe Library

[0117] Oligonucleotides are either purchased from vendors such asGenetic Designs, Inc. Houston, Tex. or made on an Applied Biosystems381A DNA synthesizer. The probe library ia synthesized using nucleotideor nucleotide analogs that have been covalently modified with aconductive polymer, with each probe incorporating a unique conductivepolymer.

[0118] Reliable hybridizations are obtained with probes six to eightnucleotides in length. These procedures allow maximal reduction in thenumber of probes per library, reducing the costs of sequencing reaction.A complete library of 4,096 random hexamers, each labeled with a uniqueconductive polymer label is synthesized as described above. Labelmoieties are covalently attached to a nucleotide precursor prior toprobe synthesis. Each probe is labeled at the 3′ end with a single labelmoiety.

Example 2 Hybridization Procedures

[0119] Target DNA is immobilized by optical trapping in an input chamberof a microfluidic device. The input chamber is filled with ahybridization solution (0.5M Na2 HPO4, pH 7.2, 7% sodium lauroylsarcosine). A hexamer probe library as described above is added andallowed by hybridize to the target nucleic acid by incubation at 60° C.for 1 hr. Probe concentration is 10 μg of DNA in 100 μl of hybridizationsolution. Hybridization is stopped by the introduction of 6× SSC washingsolution and non-hybridized probes are removed by washing multipletimes. The target nucleic acid is released from the optical trap andenters the channel for detection of hybridized probes.

Example 3 Analysis of Bound Probes

[0120] The hybridized nucleic acid is released from the chamber and anelectro-osmotic motive force is applied, driving the hybridized nucleicacid down the channel past the detection unit. As each probe passes themicroelectron beam and microspectrophotometer the fluorescence of theassociated label is detected as a spectral profile, which is associatedwith a particular probe of known sequence. The linear order of probemolecules is assembled by the analysis of multiple hybridizations. Oncethe linear order of probes is obtained the target nucleic acid sequenceis be determined.

[0121] All of the COMPOSITIONS, METHODS and APPARATUS disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the COMPOSITIONS, METHODS and APPARATUS and in the steps orin the sequence of steps of the methods described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A composition comprising a probe library, thelibrary consisting of at least 256 different probes, wherein eachdifferent probe is distinguishably labeled.
 2. The composition of claim1, wherein the probes are oligonucleotides, chemically modifiedoligonucleotides, oligonucleotide analogs or peptide nucleic acids. 3.The composition of claim 1, wherein the probes are labeled withconductive polymers.
 4. The composition of claim 1, wherein the libraryconsists of about 500, about 1000, about 2000, about 4000, about 8000,about 16,000, about 32,000 or about 64,000 different probes.
 5. Thecomposition of claim 1, wherein the probes comprise random sequences. 6.The composition of claim 5, wherein the random sequences are 4, 5, 6, 7or 8 bases long.
 7. The composition of claim 5, wherein the randomsequences are attached to one or more constant sequences.
 8. Thecomposition of claim 7, wherein the constant sequences are 1, 2, 3 or 4bases long.
 9. A method of sequencing a target nucleic acid comprising:a) obtaining a target nucleic acid; b) hybridizing the nucleic acid to aprobe library, the library consisting of at least 256 different probes,wherein each different probe is distinguishably labeled; c) separatingthe hybridized nucleic acid from unhybridized probes; and d) detectingthe order of hybridized probes along the nucleic acid.
 10. The method ofclaim 9, further comprising moving the hybridized nucleic acid past adetection unit, wherein the nucleic acid is in an approximately linearconformation.
 11. The method of claim 9, wherein the probes are labeledwith conductive polymers.
 12. The method of claim 11, wherein the probelabels are excited with an electron beam.
 13. The method of claim 12,wherein the excited probe labels emit a fluorescent signal.
 14. Themethod of claim 13, wherein the fluorescence emission spectra of theprobe labels are used to detect the order of hybridized probes along thenucleic acid.
 15. The method of claim 10, wherein the hybridized nucleicacid moves past the detection unit in a microchannel or amicrocapillary.
 16. The method of claim 9, wherein a single targetnucleic acid molecule is sequenced at a time.
 17. The method of claim 9,wherein two or more target nucleic acid molecules are sequenced at atime.
 18. A nucleic acid sequencing apparatus comprising: a) an inputchamber; b) a microchannel in fluid communication with the inputchamber; and c) a detection unit operably coupled to the microchannel,the detection unit comprising an electron beam source and a detector.19. The apparatus of claim 18, further comprising an informationprocessing and control system operably coupled to the detector.
 20. Theapparatus of claim 18, further comprising a fluidic focusing regionbetween the input chamber and the microchannel.
 21. The apparatus ofclaim 18, further comprising a target nucleic acid hybridized tomultiple labeled probes, each probe distinguishably labeled.
 22. Theapparatus of claim 21, wherein the probes are labeled with conductivepolymer molecules.
 23. The apparatus of claim 20, wherein the fluidicfocusing region contains a separating comb.
 24. The apparatus of claim18, wherein the electron beam source is a micro column electron beamsource.
 25. The apparatus of claim 24, wherein the micro column electronbeam source is as shown in FIG.
 5. 26. The apparatus of claim 18,further comprising an array of microfluidic sequencing units.